EPITHELIAL H+ TRANSPORT--STRUCTURE OF H,K-ATPASE

本工作的长期目标是了解H+的机制 哺乳动物胃粘膜壁细胞的分泌。 的 胃溃疡的病理生理与H，K-ATP酶密切相关 活性，奥美拉唑直接抑制，H2间接抑制 对手。 上皮细胞中H+转运的一个鲜为人知的方面是 ATP的标量能量转化为 矢量能量表现为H+梯度。 H，K-ATPase是一种独特的 这种机制的实验模型，很容易从 胃粘膜，在那里它形成了一百万倍的H+梯度穿过胃粘膜， 胃上皮 H，K-ATP酶cDNA的克隆和序列分析表明， 泵的主要结构。 亲水性分析预测多个 跨膜α-螺旋，其可想象地形成H+和K+孔。 实验证实或反驳重要的建模这些 机制是了解H，K-ATP酶的构象变化发生 在运输过程中。 在本研究中，H，K-ATP酶的定位在 壁细胞顶膜将用单克隆抗体探测 （Mab）。 免疫电镜观察单克隆抗体的侧性。 表位 H，K-ATP酶的结构域。 配体诱导（E1-E2）和刺激诱导 构象转换将使用Mab，胃囊泡， 和分离的壁细胞。 该研究将为H+提供直接证据 泵的拓扑结构，将阐明构象转换至关重要， 运输，将有助于解释H，K-ATP酶电子 衍射数据，将告知定点诱变，并将 有助于理解H+运输的分子机制。

The long term goal of this work is to understand the mechanism of H+ secretion by parietal cells of mammalian gastric mucosa. The pathophysiology of gastric ulcers is intimately related to H,K-ATPase activity, which is inhibited directly by omeprazole and indirectly by H2 antagonists. A poorly understood aspect of H+ transport in epithelia is the mechanisms by which the scalar energy of ATP is converted into vectorial energy manifested in a H+ gradient. The H,K-ATPase is a unique experimental model for this mechanism, being readily isolated from the gastric mucosa, where it forms a million fold gradient of H+ across the gastric epithelium. Cloning and sequencing of H,K-ATPase cDNA has revealed the primary structure of the pump. Hydropathy analysis predicts multiple transmembrane alpha-helices which conceivably form H+ and K+ pores. Experimental confirmation or refutation important in modelling these mechanisms is knowledge of H,K-ATPase conformational changes occurring during transport. In this study, the orientation of the H,K-ATPase in the parietal cell apical membrane will be probed with monoclonal antibodies (Mab). The sidedness of Mab and by immunoelectron microscopy. Epitopic domains of the H,K-ATPase. Ligand-induced (E1-E2) and stimulation-induced conformational transitions will be documented using Mab, gastric vesicles, and isolated parietal cells. The study will provide direct evidence for H+ pump topography, will clarify conformational transitions essential to transport, will aid in the interpretation of H,K-ATPase electron diffraction data, will inform site-directed mutagenesis, and will contribute to understanding of the molecular mechanism of H+ transport.